In-vitro Antimicrobial Activities of Some Iranian Conifers.

Male and female leaves and fruits of eleven different taxons of Iranian conifers (Cupressus sempervirens var. horizontalis, C. sempervirens var. sempervirens, C. sempervirens cv. Cereifeormis, Juniperus communis subsp. hemisphaerica, J. excelsa subsp. excelsa, J. excelsa subsp. polycarpos, J. foetidissima, J. oblonga, J. sabina, Platycladus orientalis and Taxus baccata) were collected from different localities of Iran, dried and extracted with methanol. The extracts were tested for their antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Candida albicans. The extracts were screened qualitatively using four different methods, the disc diffusion, hole plate, cylinder agar diffusion and agar dilution methods, whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method. The best result was obtained by means of hole plate method in qualitative determination of antimicrobial activities of extracts and the greatest activity was found against S. aureus in all tested methods.


Introduction
Iranian conifers consist of two families: Cupressaceae and Taxaceae. Cupressaceae consists of one species of Cupressus and one species of Platycladus. The Taxaceae consists of only one species of Taxus. C. sempervirens, P. orientalisand J. excelsasubspexcelsaare monoecious and others are diecious (1-3).
The progressive resistance of human pathogen microbes against antibiotics, such as methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP) and vancomycin-resistant Enterococci (VRE), is a growing problem and it is therefore extremely important to find out and develop new antimicrobial compounds. The screening of plant extracts for their antimicrobial activity has shown that higher plants represent a potential source of new antiinfective compounds (9).
The antimicrobial efficacy of obtained oils from different species of Iranian conifers was showed previously (10-114).

Extraction of the samples
Individual fresh leaves of male and female of each plant as well as fruits of them (100 g fresh wt.) were ground by a blinder. Each sample was macerated in pure methanol for 24 h. The samples were then extracted using a percolator. The extracted solutions (27 samples) were concentrated to dryness at 50°C under reduced pressure. The methanol extracts of leaves and fruits of each taxon were evaluated for their antimicrobial activity.

Isolation and Quantification of alkaloids, flavonoids, saponins and tannins
The fruits and leaves of each plant (500 g) were dried at 50°C and then powdered separately. Each powder was defatted with petroleum ether (bp 40-60°C) using Soxhlet apparatus (6 h). The chemical components of defatted powders were extracted by maceration with methanol (four times). The methanol extracts were concentrated at reduced pressure and the presence of alkaloids (16), flavonoids (17), saponins (18) and tannins (19) were determined ( Table 2).
The purpose of this study is to investigate potential antimicrobial activity of methanol extracts of Iranian conifers, by means of the hole plate, cylinder and disc agar diffusion and agar dilution methods in order to compare the suitability of the screening methods. Whereas the minimum inhibitory concentrations (MIC) of each extract were determined by the agar dilution method.

Plant material
Plant specimens were collected from different parts of the country (Table 1). The plants were identified by Dr. M. Assadi, Research Institute of Forest and Rangelands, Ministry of Jahad Keshavarzi, Iran, Voucher specimens of the taxa have been deposited in the Herbarium of National Botanical Garden of Iran (TARI).
The collected materials were stored at -20°C in order to avoid unfavorable changes in the chemical components (15).

Hole diffusion method
This assay was performed using a suspension with 0.5 McFarland standard turbidity. Holes of 6 mm diameter were then made on the Mueller Hinton agar (Merck) plate (8 mm thick) inoculated by flooding and filled with 50 µL of methanol extract. The plates containing the bacteria and C. albicans were respectively incubated at 37°C and 25°C for 24 and 48 h. The antimicrobial activity was evaluated by measuring the inhibition zone (IZ) around each hole. They were recorded as (-) for non-active samples and (+) for samples presenting IZ greater than 6 mm (the diameter of the hole) (20).

Cylinder plate diffusion test
This method is the same as hole diffusion test except that the filled cylinder containing 200 µL of different concentration of each extract used as the filled holes. The negative and positive controls were methanol and solutions of two antibiotics (gentamycin 0.2 µg/mL and clotrimazole 0.16 µg/mL), respectively (21).

Disc diffusion method
This assay was performed using the filter paper disc diffusion method on Mueller Hinton and sabouraud dextrose agar respectively for bacteria and C. albicans. The plates were incubated under sterile conditions at 37°C for 24 h. Inoculums were prepared using a suspension with 0.5 McFarland standard turbidity and the culture was spread over the plates by means of a sterile cotton swab.
Plant extracts (0.25-2 mg/disc) were prepared and placed on plates earlier inoculated with microbial suspension. This was done to evaluate the sensitivity of the extracts at which microbial growth was inhibited effectively (22).

Agar well dilution test
Briefly, the methanol extracts (100, 50, 25 and 12.5 µg/mL) were diluted in molten Mueller Hinton agar (MHA, Merck) on 24 well plates. All bacterial strains were grown in Mueller Hinton broth (MHB, Merck) for 4 h at 37°C. Bacterial suspensions with 0.5 McFarland standard turbidity (≈10 8 cfu/ mL), were prepared by dilution with Mueller Hinton broth. The diluted inoculums were added to a Steer's replicator calibrated and incubated for 24 h at 37°C and 48 h at 25°C respectively for bacteria and C. albicans (23).

Results and Discussion
The antimicrobial activities of 27 methanol extracts of different parts of Iranian conifers were determined using four different methods. Screening was carried out at four different concentrations against S. aureus, E. coli, P. aeruginosa and C. albicans strains to examine the sensitivity against the mentioned microorganism. Table 3 shows the most sensitive microbial strains in different methods.  Table 3. Most sensitive microbial strains in different methods. Table 4 shows the antimicrobial activity of Iranian connifer extracts (25, 50, 100 and 200 mg/mL) in three different methods. Methanol extracts exhibited only weak or no activity in cylinder agar diffusion method perhaps because of the low extracts diffusion in agar or may be the precipitation of insoluble material that inhibits further diffusion of active constituent.       Table 4. Antimicrobial activity of Iranian connifer extracts for concentration of 25, 50, 100 and 200 g L -1 in three different methods. (-) 9.3 ± 0.26 6.26 ± 0.15
The micro-organism sensitivity in low (25 µg/mL) and high (200 µg/mL) concentrations of extracts were differing in hole plate method as shown in Table 5.

MIC of each extracts as determined by agar diffusion method
The MIC exhibited by the Iranian conifer extracts against tested bacterial and fungal strains are given in et al. (24,25) reported that extracts of C. sempervirens: showed no activity against tested strains. The contradictory about S. aurus might be due to the extract concentrations, microbial strains or solvent used in our study. Methanol extracts had the most marked antimicrobial effects of all the tested species in different studies (26). C. sempervirens and C. sempervirens. cv. Cereiformis have been found to possess antimicrobial activities against P. aeroginosa in our study. It is believed that this effect shown by these taxa is due to the limitations discussed above. Essential oil of C. sempervirens was shown to be a potent inhibitor of S. aurus and P. aeroginosa, (27) therefore, the presence of essential oil constituent in extracts can be assumed. Since it is known that the leaves and fruits of cupressus species contain Camphen, Quercetin, Catechin, Linalool, Borneol and Sabinen, a part of the antimicrobial activities we investigated might be due to these constituents (28-30).

Antimicrobial effects of the investigated Juniperus species
As Dornberger et al. (31) had found antimicrobial properties in J. communis, and Muhammad et al. and Topcu et al. (32,33) found evidence of antibacterial activity from the leaves and fruit of J. excelsa, it was not a surprise to These results indicate that the essential oils derived from coniferous trees, which have mild antimicrobial properties, can inhibit the growth of Gram-positive and Gram-negative bacteria and fungi. (1)